Apoptosis is the process of programmed cell death (PCD) that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in an extensive variety of diseases. Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.
Caspases are proteins that are highly conserved, cysteine-dependent, aspartate-specific proteases. There are two types of caspases: initiator caspases, caspase 2, 8, 9, and 10, and effector caspases, caspase 3, 6 and 7. The activation of initiator caspases requires binding to specific oligomeric adaptor protein. Effector caspases are then activated by these active initiator caspases through proteolytic cleavage. The active effector caspases then proteolytically degrade a host of intracellular proteins to carry out the cell death program.
Detection and measurement of apoptosis has been found to be useful in detecting the presence or severity of diseases. Cytokeratins (CK) are known to be useful markers for many different diagnostic methods. For example, circulating fragments of cytokeratin 18 (also CK18, CK-18, K18, or K-18), a cytoskeletal marker of cell death, such as hepatocyte death, have been shown in several studies to indicate the transition from benign fatty liver to nonalcoholic steatohepatitis (NASH), with a risk of fibrosis, in patients with nonalcoholic fatty liver disease (NAFLD).
NAFLD is a spectrum of disorders characterized by hepatic steatosis, which may be benign (nonalcoholic fatty liver or NAFL) or which may progress via inflammation and fibrosis to NASH followed by cirrhosis and liver failure. Liver biopsy, the standard diagnostic approach for NAFL/NASH, has limitations due to sampling site variability, cost and procedure-related morbidity. Appropriate circulating biomarkers may enable diagnosis, staging and monitoring of NAFL/NASH with fewer biopsies.
Assays incorporating CK18 as a biomarker may be developed to monitor the severity of such diseases by measuring apoptosis. CK18 is also useful for detecting and monitoring other liver diseases including hepatitis, biliary sclerosis, and poisoning. CK18 is also useful for quantifying cell death in cancer and diseases that are part of the metabolic syndrome, including degenerative diseases, such as cardiovascular and liver disease.
This disclosure covers antibodies and related assays and methods for detecting cell death by measuring the presence of CK18 fragments, that are typically present only when a cell is dying and after full-length CK18 is exposed to and cleaved by caspases.